The references cited herein are not admitted to be prior art to the claimed invention.
Neuropeptides present in the hypothalamus play a major role in mediating the control of body weight. (Flier, et al., 1998. Cell, 92, 437-440.) Melanin-concentrating hormone (MCH) is a cyclic 19-amino acid neuropeptide synthesized as part of a larger pre-prohormone precursor in the hypothalamus which also encodes neuropeptides NEI and NGE. (Nahon, et al., 1990. Mol. Endocrinol. 4, 632-637.) MCH was first identified in salmon pituitary, and in fish MCH affects melanin aggregation thus affecting skin pigmentation. In trout and in eels MCH has also been shown to be involved in stress induced or CRF-stimulated ACTH release. (Kawauchi, et al., 1983. Nature 305, 321-323.)
In humans two genes encoding MCH have been identified that are expressed in the brain. (Breton, et al., 1993. Mol. Brain Res. 18, 297-310.) In mammals MCH has been localized primarily to neuronal cell bodies of the hypothalamus which are implicated in the control of food intake, including perikarya of the lateral hypothalamus and zona inertia. (Knigge, et al., 1996. Peptides 17, 1063-1073.)
Pharmacological and genetic evidence suggest that the primary mode of MCH action is to promote feeding (orexigenic). MCH mRNA is up regulated in fasted mice and rats, in the ob/ob mouse and in mice with targeted disruption in the gene for neuropeptide Y (NPY). (Qu, et al., 1996. Nature 380, 243-247, and Erickson, et al., 1996. Nature 381, 415-418.) Injection of MCH centrally (ICV) stimulates food intake and MCH antagonizes the hypophagic effects seen with xcex1 melanocyte stimulating hormone (xcex1MSH). (Qu, et al., 1996. Nature 380, 243-247.) MCH deficient mice are lean, hypophagic and have increased metabolic rate. (Shimada, et al., 1998. Nature 396, 670-673.)
MCH action is not limited to modulation of food intake as effects on the hypothalamic-pituitary-axis have been reported. (Nahon, 1994. Critical Rev. in Neurobiol. 8, 221-262.) MCH may be involved in the body response to stress as MCH can modulate the stress-induced release of CRF from the hypothalamus and ACTH from the pituitary. In addition, MCH neuronal systems may be involved in reproductive or maternal function.
Several references describe a receptor that is indicated to bind MCH (xe2x80x9cMCH-R1xe2x80x9d). (Chambers, et al., 1999. Nature 400, 261-265; Saito, et al., 1999. Nature 400, 265-269; Bxc3xa4chner, et al., 1999. FEBS Letters 457:522-524; and Shimomura, et al., 1999. Biochemical and Biophysical Research Communications 261, 622-626.)
The present invention features HG67 nucleic acids and HG67 polypeptides. HG67, also referred to herein as xe2x80x9cMCH-R2xe2x80x9d, is a G-protein coupled receptor having a high degree of sequence identity with MCH-R1. The amino acid sequence for HG67 is provided by SEQ. ID. NO. 1. The cDNA sequence of HG67 is provided by SEQ. ID. NO. 2.
HG67 polypeptides contain a region of at least 9 contiguous amino acids that is present in SEQ. ID. NO. 1. Such polypeptides may contain additional regions present, or not present, in SEQ. ID. NO. 1. HG67 polypeptides include, for example, full length HG67, HG67 fragments, and chimeric polypeptides containing all or a portion of HG67 along with amino acid region(s) not from HG67.
HG67 nucleic acids contain a region that encodes for a HG67 polypeptide or contains at least 18 contiguous nucleotides that is present in SEQ. ID. NO. 2 or the complement thereof. Such nucleic acid may contain additional regions present, or not present, in nucleic acid encoding for HG67 or present in SEQ. ID. NO. 2 or the complement thereof. HG67 nucleic acids include, for example, nucleic acid encoding for all or a portion of HG67, nucleic acid containing all or a portion of SEQ. ID. NO. 2, and recombinant nucleic acid encoding all or a portion of HG67 and/or containing all or a portion of SEQ. ID. NO. 2.
Thus, a first aspect of the present invention describes a purified HG67 polypeptide. The polypeptide comprises at least 9 contiguous amino acids of SEQ. ID. NO. 1.
A xe2x80x9cpurified polypeptidexe2x80x9d represents at least 10% of the total protein present in a sample or preparation. In preferred embodiments, the purified polypeptide represents at least about 50%, at least about 75%, or at least about 95% of the total protein in a sample or preparation. Reference to xe2x80x9cpurified polypeptidexe2x80x9d does not require that the polypeptide has undergone any purification and may include, for example, chemically synthesized polypeptide that has not been purified.
Another aspect of the present invention describes a purified HG67 nucleic acid. The nucleic acid comprises either (a) a region of at least 18 contiguous bases present in either bases 1-180 or 396-1020 of SEQ. ID. NO. 2, or the complement thereof; or (b) a region encoding for at least 9 contiguous amino acids present in either bases 1-60 or 150-340 of SEQ. ID. NO. 1. Reference to the presence of one region does not indicate that another region is not present. For example, in different embodiments the nucleic acid can comprise or consist of a nucleic acid encoding for SEQ. ID. NO. 1 and can comprise or consist of the nucleic acid sequence of SEQ. ID. NO. 2.
A xe2x80x9cpurified nucleic acidxe2x80x9d represents at least 10% of the total nucleic acid present in a sample or preparation. In preferred embodiments, the purified nucleic acid represents at least about 50%, at least about 75%, or at least about 95% of the total nucleic acid in a sample or preparation. Reference to xe2x80x9cpurified nucleic acidxe2x80x9d does not require that the nucleic acid has undergone any purification and may include, for example, chemically synthesized nucleic acid that has not been purified.
Another aspect of the present invention describes an expression vector. The expression vector comprises a nucleotide sequence encoding for at least 9 contiguous amino acids provided in SEQ. ID. NO. 1, wherein the nucleotide sequence is transcriptionally coupled to an exogenous promoter. Reference to a nucleotide sequence xe2x80x9ctranscriptionally coupled to an exogenous promoterxe2x80x9d indicates the presence and positioning of an RNA promoter such that it can mediate transcription of the nucleotide sequence and that the promoter is not naturally associated with the nucleotide sequence.
Another aspect of the present invention describes a recombinant cell comprising an expression vector encoding for a region of at least 9 contiguous amino acids of SEQ. ID. NO. 1. The expression vector contains a promoter that is transcriptionally coupled to nucleic acid encoding for the region and is recognized by an RNA polymerase present in the cell.
Another aspect of the present invention describes a recombinant cell made by a process comprising the step of introducing into the cell an expression vector encoding for a region of at least 9 contiguous amino acids of SEQ. ID. NO. 1. Preferably, the expression vector contains a promoter that is transcriptionally coupled to nucleic acid encoding for the region and is recognized by an RNA polymerase present in the cell. The expression vector can be used to insert recombinant nucleic acid into the host genome or can exist as an autonomous piece of nucleic acid.
Another aspect of the present invention features a purified antibody preparation comprising an antibody that binds to HG67. A xe2x80x9cpurified antibody preparationxe2x80x9d is a preparation where at least 10% of the antibodies present bind to HG67. In preferred embodiments, antibodies binding to HG67 represent at least about 50%, at least about 75%, or at least about 95% of the total antibodies present. Reference to xe2x80x9cpurified antibody preparationxe2x80x9d does not require that the antibodies in the preparation have undergone any purification.
Another aspect of the present invention describes a method of producing a polypeptide comprising at least 9 contiguous amino acids of SEQ. ID. NO. 1. The method involves the step of growing a recombinant cell able to express the polypeptide from an expression vector.
Another aspect of the present invention describes a method for screening for a compound able to bind to HG67. The method involves the following steps: (a) expressing a polypeptide comprising at least about 9 contiguous amino acids of SEQ. ID. NO. 1 from recombinant nucleic acid; (b) providing to the polypeptide a test preparation comprising one or more test compounds; and (c) measuring the ability of the test preparation to bind to the polypeptide.
A test preparation contains one or more compounds being tested. In different embodiments a test preparation contains 10 or more compounds, 5 or more compounds, or 1 compound.
A xe2x80x9crecombinant nucleic acidxe2x80x9d is a nucleic acid containing one or more regions not naturally associated with each other. Examples of recombinant nucleic acid include an HG67 region present on a nucleic acid also containing one or more regulatory elements not naturally associated with the HG67 region, viral elements, or selectable markers.
Another aspect of the present invention describes a method for screening for HG67 activity. The method involves (a) contacting a cell expressing a recombinant nucleic acid encoding for a G-protein coupled receptor comprising at least 9 contiguous bases of SEQ. ID. NO. 1 with a test preparation comprising one or more test compounds; and (b) measuring the effect of the test preparation on the activity of the receptor.
Another aspect of the present invention describes a method for achieving a beneficial effect in a patient comprising the step of modulating HG67 activity in the patient. Preferably, HG67 activity is modulated by causing a decrease in the activity or expression of the receptor. HG67 activity can be modulated using, for example, organic compounds active at the HG67 receptor and nucleic acid able to decrease HG67 expression.
Another aspect of the present invention describes a method for screening for a compound able to bind HG67 or a fragment thereof. The method comprising the steps of: (a) expressing a polypeptide comprising the amino acid sequence of SEQ. ID. NO. 1 or fragment thereof from recombinant nucleic acid; (b) providing to the polypeptide a labeled MCH ligand and a test preparation comprising one or more test compounds; and (c) measuring the effect of the test preparation on binding of the labeled MCH ligand to the polypeptide.
A xe2x80x9cMCH ligandxe2x80x9d refers to a polypeptide that binds to HG67 at the MCH binding site. MCH ligands include human MCH, salmon MCH, and derivatives thereof.
Another aspect of the present invention describes a method for screening for a compound able to modulate HG67 activity. The method comprises the steps of: (a) contacting a cell line expressing recombinant nucleic acid encoding for a polypeptide comprising (i) the amino acid sequence of SEQ. ID. NO. 1 or a fragment thereof that binds MCH and (ii) a region that functionally couples to a G protein, wherein the polypeptide is able to transduce an intracellular signal upon MCH ligand binding, with a MCH ligand and a test preparation comprising one or more test compounds; and (b) measuring the effect of the preparation on polypeptide activity.
Other features and advantages of the present invention are apparent from the additional descriptions provided herein including the different examples. The provided examples illustrate different components and methodology useful in practicing the present invention. The examples do not limit the claimed invention. Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present invention.